Methods of screening apoptosis modulating compounds, compounds identified by said methods and use of said compounds as therapeutic agents

ABSTRACT

A method of screening cellular polypeptides for pro-apoptotic or anti-apoptotic activity in a cell of a particular cell-type, said method comprising: (a) culturing cells of said particular cell-type under non apoptotic conditions and culturing cells of said particular cell-type under apoptotic conditions, and (b) determining subcellular localisation of said cellular polypeptides in the cultured cells, wherein a localization of a cellular polypeptide in lipid rafts in cultured cells under non apoptotic conditions and a segregation of said cellular polypeptide from lipid rafts in cultured cells under apoptotic conditions is indicative that said cellular polypeptide has a pro-apoptotic or an anti-apoptotic activity in said particular cell-type.

The invention relates to the modulation of apoptosis in mammalian cells.More particularly, the invention provides methods for identifying novelpro-apoptotic or anti-apoptotic cellular polypeptides, methods ofscreening compounds which modulate apoptosis, and method of detectingearly events of the apoptotic process.

Apoptosis or programmed cell death is an active process in which cellsinduce their self-destruction in response to specific cell death signalsor in the absence of cell survival signals. This active process isactually essential in the normal development and homeostasis ofmulticellular organisms. It is opposed to necrosis which is cell deathoccurring as a is result of severe injurious changes in the environment.

Apoptosis of a cell can be characterized at least by

-   -   the rapid condensation of the cell with collapse of the nucleus        but preservation of membranes; or,    -   cleavage of nuclear DNA at the linker regions between        nucleosomes to produce fragments which can be easily visualized        by agarose gel electrophoresis as a characteristic ladder        pattern.

Various pathologies occur due to a defective or aberrant regulation ofapoptosis in the affected cells of an organism. For example, defectsthat result in a decreased level of apoptosis in a tissue as compared tothe normal level required to maintain the steady-state of the tissue canpromote an abnormal increase of the amount of cells in a tissue. Thishas been observed in various cancers, where the formation of tumorsoccurs because the cells are not dying at their normal rate. Some DNAviruses such Epstein-Barr virus, African swine fever virus andadenovirus, also inhibit or modulate apoptosis, thereby repressing celldeath and allowing the host cell to continue reproducing the virus.

On the contrary, a defect resulting in an increase of cell death in atissue may be associated with degenerative disorders wherein cells aredying at a higher rate than they regenerate. This is observed in variousdisorders, such as AIDS, senescence, and neurodegenerative diseases.

Compounds that modulate positively or negatively apoptosis can providemeans for the treatment or the prevention of these disorders. As aconsequence, the delineation of apoptotic pathways provides targets forthe development of therapeutic agents that can be used to modulate theresponse of a cell to apoptotic or cell survival signals.

Progresses have been made in identifying extracellular, intracellularand cell surface molecules that regulate apoptosis. Previous studieshave focused on the identification of specific cell death signals, (suchas the deprivation of growth factors, the FAS/TNF system, genotoxicagents, glucocorticoïds . . . ), members of the Bcl-2 family andICE-type proteases. But critical steps in apoptotic pathways remain tobe identified.

Accordingly, there is still a need in identifying the cellularmechanisms involved in apoptotic pathways, and target for thedevelopment of therapeutic agents that can be used to modulate cellapoptosis.

Among the different transducing agents, the Bcl-2 family proteins act asan intracellular checkpoint in the apoptotic pathway. The family isdivided into two functional groups (médecine/sciences 97;13:384-6): theproteins that suppress cell-death (anti-apoptotic members such as Bcl-2,Bcl-x_(L), Bcl-w, Bag-1, McI-1, A1) and the proteins that promote celldeath (pro-apoptotic members such as Bim, Nix, Hzk, Bax, Bak, Bcl-x_(s),Bad, Bik). The Bcl-2 family has been defined by sequence homology basedupon specific conserved motifs termed BCL-homology regions (BH1, BH2,BH3 and BH4 domains). BH1, BH2 and BH3 domains have been shown to beimportant in homodimerization or heterodimerization and in modulatingapoptosis. Anti-apoptotic molecules have a specific BH4 domain.

It has been proposed that the ratio of pro-apoptotic members toanti-apoptotic members expressed in a cell determines whether this cellwill respond to an apoptotic signal. Indeed, pro-apoptotic andanti-apoptotic members antagonized each other by forming inactiveheterodimers (Oltvai et al., 1993, Cell 74: 609-619), as a consequenceonly the balance may promote or prevent a cell to undergo apoptosis.

More recently, it has been shown that phosphorylation of Bcl-2 proteinscan also modulate their activity. Indeed, different anti-apoptoticpathways are likely to be activated by growth factors, involvingphosphatidylinositol 3 kinase (Pl3K), Akt kinase and Ras activatedkinases.

In particular, upon stimulation of cells with IL-3 and NGF, thepro-apoptotic Bad protein (Bcl-x_(L)/Bcl-2 Associated cell Deathregulator, Downward, 1999, Nature Cell Biol 1: 33-35) becomes serinephosphorylated, resulting in association to 14-3-3 protein (Hsu et aL,1997, Mol Endocrinol 11: 1858-1867). It was proposed that suchinteraction facilitates the translocation of phosphorylated Bad from themitochondrial membrane to cytosolic compartments, sequestering ittherein and thus, preventing further interaction with otheranti-apoptotic Bcl-2 members (U.S. Pat. No. 5,856,445).

It was further shown that association of 14-3-3 protein to Bad isdependent upon serine 155 phosphorylation of Bad (WO 0110888, ApoptosisTechnology Inc., 2001).

The results disclosed in the present invention indicate that some pro-or anti-apoptotic proteins especially of the Bcl-2 family, are regulatedthrough a newly identified subcellular localization that is in lipidrafts formed in the plasma membrane. This observation offers a way to anovel general mechanism of regulation of cell apoptosis that may play arole in the regulation of pro- or anti-apoptotic molecules in responseto cell death or cell survival signals.

Localization of proteins to distinct subcellular compartments, includingmembranes, is a critical event in multiple cellular pathways.

Plasma membranes of many cell types contain microdomains commonlyreferred to as lipid rafts, which are biochemically distinct from bulkplasma membranes (Brown and London, 1998, Annu Rev Cell Dev Biol 14:111-136). These domains consist of dynamic assemblies of sphingolipidsand cholesterol. More specifically, the presence of saturatedhydrocarbon chains in sphingolipids allows for cholesterol to be tightlyintercalated, leading to the presence of distinct liquid-ordered phases,and thereby more fluid, lipid bilayer. Lipid rafts can be isolated bysubcellular fractionation and density gradient ultracentrifugationaccording to methods described in Hacki et al. (Oncogene 2000 19:2286-2295) and Millan et al. (Eur J Immunol 1998 28: 2675-3684). Theycan also be visualized in intact cells by confocal microscopy using, forexample, fluorescently labelled cholera toxin subunit B (CTx) whichbinds to the ganglioside GM1 (Harder et aL, 1998, J Cell Biol 141:929-942). One key element of lipid rafts is that they can include orexclude proteins to varying degrees.

In T cells, a number of proteins involved in signal transduction such asIcK, Lat, copurify with lipid rafts isolated on sucrose gradient.Disruption of rafts integrity by a variety of methods inhibits earlyactivation events, supporting a critical role for these domains in therecruitment for signalling and thus, in signal transduction from cellsurface receptors. For example, the antitumor ether lipid1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3;edelfosine) was shown to trigger apoptosis via translocation of Fas tolipid rafts and subsequent Fas recruitment by lipid rafts (Gajate andMollinedo, Blood, 2001, 98: 3860-3863).

The present invention results from the discovery of a novel mechanism ofcellular regulation of the activity of pro or anti-apoptotic moleculesin a cell by translocation of these molecules into lipid rafts under nonapoptotic conditions such as proliferative conditions or conditionswhere cells do not divide.

Indeed, the inventors have surprisingly found that interaction of apro-apoptotic protein, such as the Bad protein, with rafts is an activeprocess regulated by cytokines or growth factors. They have also shownthat segregation of this molecule from rafts in cytokine or growthfactor deprived-cells is involved in the induction of apoptosis andassociated with raft disorganization.

The invention thus provides methods for identifying cellularpolypeptides which have pro-apoptotic or anti-apoptotic activity in aparticular cell type.

The invention also provides means for screening apoptosis modulatingcompounds which interfere with the newly identified mechanism ofapoptosis regulation. Candidate compounds in this respect may eitherinterfere by blocking, preventing or stimulating translocation of one orseveral pro- or anti-apoptotic polypeptides in lipid rafts under nonapoptotic conditions. Candidate compounds may in addition oralternatively interfere by disrupting or reconstituting lipid rafts in acell which normally produce pro- or anti-apoptotic polypeptides locatedin lipid rafts under non apoptotic conditions. According to anotherembodiment, candidate compounds may in addition or alternativelyinterfere by segregation of pro- or anti-apoptotic polypeptides fromlipid rafts.

The invention also provides a compound capable of modulating associationof a pro- or anti-apoptotic polypeptide with lipid rafts.

The invention also provides a compound capable of modulating transfer ofa pro- or anti-apoptotic polypeptide between a lipid raft and anothercellular localization.

The invention also provides for the use of compounds capable ofmodulating lipid rafts formation or of modulating translocation of pro-or anti-apoptotic proteins in rafts in the preparation of a medicine forthe prevention and/or treatment of disorders induced by or associatedwith a defective regulation of cell death as well as of specificpathologies in which the death of infected or deregulated cells may beat least art of a therapy.

Among the several advantages of the present methods, it should be notedthat the apoptotic or non apoptotic state of a cell can be determinedaccording to the present methods in a relatively short period of time byanalysing lipid raft organization. In particular, there is no need toquantify specific gene expression. The methods of the invention are thusparticularly appropriate for routine high throughput screening ofapoptosis modulating compounds.

Furthermore, the invention provides methods for detecting early eventsof the apoptotic process in a cell.

A first object of the invention is a method of screening cellularpolypeptides for pro-apoptotic or anti-apoptotic activity in a cell of aparticular cell-type, said method comprising:

-   -   a. culturing cells of said particular cell-type under non        apoptotic conditions and culturing cells of said particular        cell-type under apoptotic conditions; and,    -   b. determining subcellular localisation of said cellular        polypeptides in the cultured mammalian cells;        wherein a localization of a cellular polypeptide in lipid rafts        in cells cultured under non apoptotic conditions and a        segregation of said cellular polypeptide from lipid rafts in        cells cultured under apoptotic conditions is indicative of the        pro-apoptotic or an anti-apoptotic activity of said cellular        polypeptide in said particular cell-type.

In a particular embodiment, said cultured cells are mammalian cells.

In a preferred embodiment, the cells cultured under non apoptoticconditions are cultured under proliferative conditions. According to themethods of the invention, cells are considered to be cultured “under nonapoptotic conditions” when the proportion of cells undergoing apoptoticprocess in the cell culture is relatively stable in time and does notrepresent more than 10%, preferably, more than 1% of the whole cellpopulation (depending upon the cell-type).

In a preferred embodiment, non apoptotic conditions are proliferativeconditions.

On the contrary, cells are considered to be cultured “under apoptoticconditions” when the proportion of the cells undergoing apoptoticprocess increases dramatically in time to reach, after a certain period,especially for around 24 hours, from deprivation of growth orproliferation factor or by use of an apoptotic factor, more than 50% ofthe whole cell population.

Cells which have undergone apoptotic process can be characterized, forexample, by specific cleavage of nuclear DNA which can be visualized onagarose gel electrophoresis.

As used herein, the term “cellular polypeptide” refers to anypolypeptide which is produced in a cell by gene expression. It can be apolypeptide naturally encoded in said cell especially by a native gene,or a polypeptide not naturally encoded in said cell, meaning that thegene encoding said polypeptide or a coding sequence derived from saididentified gene has been recombined in the genome of the cell to obtainexpression. It can be a mutated form of a naturally occurringpolypeptide and more specifically, a mutated form wherein the mutationis involved in abnormal subcellular localisation of said polypeptideunder proliferative growth conditions.

As used herein, the term “lipid rafts” refers to dynamic assemblies ofsphingolipids and cholesterol in plasma membranes forming microdomainswith distinct liquid-ordered phases, said microdomains stably retainingspecific structures, such as gangliosides or polypeptides such as Lck.Lipid rafts can be biochemically isolated by subcellular fractionationand density gradient ultracentrifugation according to the methodsdescribed in Hacki et al. (Oncogene 2000 19: 2286-2295) and Millan etal. (Eur J Immunol 1998 28: 2675-3684) and in the examples below. Theycan also be visualized in intact cells by confocal microscopy using, forexample, fluorescently labelled cholera toxin subunit B (CTx) whichbinds to the ganglioside GM1 which accumulate in lipid rafts (Harder etal., 1998, J Cell Biol 141: 929-942). More specifically, subcellularlocalization of a particular polypeptide in lipid rafts is determined bydouble immunofluorescence using a labelled marker detecting lipid raftsand another labelled marker detecting the polypeptide to localize. Itcan also be determined by analysing the presence of said polypeptide insubcellular fractions containing lipid rafts.

In a particular embodiment, the method of the invention is appropriateto screen a polypeptide, whose structure is known but whose function isunknown, for a pro- or anti-apoptotic activity in a particular celltype. In particular, the one skilled in the Art can use the method ofthe invention to screen polypeptides which are suspected to be involvedin apoptosis regulation according to specific features such as specificstructural domains. The screening of cellular polypeptides is howevernot lo necessarily limited to cellular polypeptides of known structure.

Several pro-apoptotic proteins have been identified so far, however,expression pattern of these proteins may vary depending upon the celltype. The method of screening is thus also useful in determining whethera putative cellular polypeptide, known to be pro or anti-apoptotic in acertain cell-type is involved in apoptosis modulation in anothercell-type.

In a particular embodiment, the screened polypeptides belong to theBcl-2 family. As mentioned hereabove, the Bcl-2 family, members arecharacterized by sequence homology based upon specific conserved motifstermed BCL-homology regions (BH1, BH2, BH3 and BH4 domains).Accordingly, their subcellular localisation can be determined by the useof a molecule which specifically recognizes a BH domain. Such moleculesencompass for example monoclonal antibodies or polyclonal antibodiesspecifically recognizing a BH domain. One example of a molecule thatinteracts with BH3 motif in PP1a (Ayllon, et al. 2000. EMBO J.; 19:2237-2246). Examples of molecules that interact with BH4 motif arereview in Admas, J. M. and Cory, S. (1998) Science, 281, 1322). In aparticular embodiment, a molecule which specifically recognizes a BH4domain is used to screen preferably for anti-apoptotic molecules. Inanother particular embodiment, a molecule which specifically recognizesa BH3 domain is used to screen for pro- or anti-apoptotic molecules. Acombination of molecules recognizing the different BH domains can alsobe used, for example to screen for pro-apoptotic molecules which haveonly the BH3 domain.

The method of the invention is also appropriate to screen novelpolypeptides of the Bcl-2 family whose structure and function areunknown at least in part, but which can be easily isolated usingmolecular recognition of their BH domain(s). As a result, in a preferredembodiment, said screened cellular polypeptides are first isolated frombiochemically isolated lipid rafts of said mammalian cells cultured inproliferative conditions by the use of a molecule which specificallyrecognizes a BH domain. More specifically, the polypeptides present inthe isolated lipid rafts can be separated on a gel and analysed byWestern Blot analysis using an antibody which recognizes a BH domain orby similar methods of protein analysis. The polypeptides recognized by aBH domain can be isolated and antibodies which recognize each isolatedpolypeptides can be produced according to usual methods well known inthe art. The subcellular localization of one or more of the isolatedpolypeptides is then determined according to the method of the inventionusing such specific antibodies to screen for apoptotic activity.

The proteins which are associated with lipid rafts may have atransmembrane domains or have undergone post-translational modificationssuch as myristoylation. In another specific embodiment, said screenedcellular polypeptides are further isolated by the use of a moleculewhich specifically recognizes a mirystoylated polypeptide.

In a preferred embodiment, the method is carried out for screeningcellular polypeptides of a cell type characterized by the production ofBad protein, i.e. Bad⁺cell type. According to another preferredembodiment, the cells are characteristic of the immune system, and mostpreferably are T cell lines.

Indeed, it is shown, in the examples below, that the pro-apoptotic Badprotein is sequestered in lipid rafts of IL-4 stimulated T-cells andsegregates from rafts in IL-4 deprived T-cells.

More specifically, it is shown in the Example that Bad can beco-purified with lipid-rafts by subcellular fractionation and densitygradient ultracentrifugation from cells under non-apoptotic conditions,especially under proliferative conditions. These results indicate thatBad is strongly associated with lipid rafts in cells cultured under nonapoptotic conditions such as proliferative conditions.

Cellular polypeptides which physically interacts with Bad protein inisolated lipid rafts of Bad⁺cells thus constitute preferred putativepolypeptides to screen for pro- or anti-apoptotic activity. Accordingly,in a preferred embodiment, the screened cellular polypeptides areisolated from isolated lipid rafts of Bad⁺cells cultured underproliferative conditions and are selected among the polypeptides whichinteract physically with the Bad protein.

Naturally, the invention also pertains to the newly identified cellularpolypeptides having pro- or anti-apoptotic activity and their use inproviding means for modulating apoptosis in cells, such as mammaliancells, expressing these polypeptides.

It is another object of the invention to provide a method of screeningcompounds for their capacity to modulate apoptosis in cells, whichproduce pro- or anti-apoptotic polypeptides which are located in lipidrafts when said cells are cultured under non apoptotic conditions, saidmethod comprising:

-   -   a) culturing said cells in a growth medium maintaining non        apoptotic conditions;    -   b) contacting said cultured cells with a candidate compound;    -   c) determining the level of one or several pro- or        anti-apoptotic polypeptides associated to lipid rafts;    -   d) selecting the compound which interferes with the association        of one or several pro- or anti-apoptotic polypeptides with lipid        rafts, said compound having the capacity to modulate apoptosis.

A compound interferes with the association of a pro- or anti-apoptoticpolypeptide when it modifies said association, including when it altersthe chemical and/or the physical nature of said association or when itprovides or influences segregation of pro- or anti-apoptoticpolypeptides from lipid rafts, or when it prevents said association, oralso when it acts on and especially promotes disruption of lipid raftsor more generally alter constitution of lipid rafts.

The invention further relates to a method of screening compounds fortheir capacity to promote apoptosis in cells, said method comprising

-   -   a) culturing cells in a growth medium maintaining non apoptotic        conditions; wherein said cells produce a pro-apoptotic protein        which is located in lipid rafts under non apoptotic conditions        of said cells;    -   b) contacting said cultured cells with a candidate compound;        and,    -   c) determining the absence or the presence of lipid rafts in        said cultured cells;    -   d) in case of presence of lipid rafts, optionally determining        the level of pro-apoptotic protein located in the lipid rafts,        wherein the absence of lipid rafts in the plasma membrane of        cells incubated with said candidate compound or if determined,        the reduced level of pro-apoptotic protein in the rafts is        indicative that said compound promotes apoptosis.

The invention also relates to a method of screening compounds for theircapacity to inhibit or prevent apoptosis of cells, said methodcomprising:

-   -   a) culturing cells in a growth medium for maintaining        non-apoptotic conditions; wherein said cells produce a        pro-apoptotic protein which is located in lipid rafts under non        apoptotic conditions;    -   b) contacting said cells with a candidate compound;    -   c) culturing cells under apoptotic conditions; and,    -   d) determining the absence or the presence of lipid rafts;    -   e) in the case of presence of lipid rafts, optionally        determining the level of pro-apoptotic protein located in the        lipid rafts,        wherein the presence of lipid rafts in the plasma membranes of        cells incubated with said candidate compound and optionally the        maintained level of proapoptotic protein in the rafts is        indicative that said candidate compound inhibits or prevents        apoptosis.

In a preferred embodiment, the cells are cultured in a growth mediumcomprising at least a cytokine or a growth factor necessary formaintaining proliferative growth conditions and step c) of the methodcomprises depriving the cells of said cytokine or growth factornecessary for maintaining proliferative growth conditions.

As used herein, the term “compound” refers to inorganic or organicchemical or biological compounds either natural (isolated) or synthetic,and especially encompass nucleic acids, proteins, polypeptides,peptides, glycopeptides, lipids, lipoproteins and carbohydrates.

Any cells in-which pro or anti-apoptotic proteins may be translocated inlipid rafts can be used in the methods of the invention. In a preferredembodiment of the methods of the invention, cells are mammalian cells.

Mammalian cells which are used in the methods of screening compounds canbe any mammalian cells whose cell survival can be controlled by aspecific cytokine or growth factor. In preferred embodiments of theabove methods, the cultured mammalian cells are selected among thosewhich produce the Bad protein as a pro-apoptotic protein. Morespecifically, preferred mammalian cells which produce a Bad protein areselected among cells characteristic of the immune system, and morepreferably among T cells.

As used herein, the term “cytokine or growth factor” refers to anymolecule which is necessary to be present in a growth medium to preventapoptotic process of a cultured cell and/or to promote cellproliferation. Known cytokines include any interleukin. Known growthfactors include the fibroblast growth factors, bFGF, aFGF, FGF6, thehepatocyte growth factors HGF/SF, the epidermis growth factor, EGF andother characterized growth factors such as IGF-1, PDGF, LIF, VEGF, SCF,TGFb, TNFa, NGF, BMP, neuregulin, thrombopdïetin and growth hormone.Growth factors according to the invention can include also,progestagenes and derivatives thereof (progesterone), oestrogens andderivative thereof (oestradiol), androgenes (testosterone),mineralocorticoids and derivatives thereof (aldosterone), LH, LH-RH, FSHet TSH hormones, T3, T4, and retinoidic acid, calcitonine E2 andF2/alpha prostaglandins. Glucocorticoids (natural or hemisynthetic, i.e.hydrocortisone, dexamethasone, prednisolone or triamcinolone), can alsobe used.

Cells characteristic of the immune system can be advantageously culturedunder stimulation with an interleukin for maintaining proliferativegrowth conditions. In particular, IL-4, IL-2 or IL-9 interleukin can beused in this context, or a mixture thereof.

However, any available apoptosis model can be used to select the celltype and the factors for non apoptotic conditions such as the growthfactor or cytokine, used in the methods. As used herein, the term“apoptosis modef” comprises any teaching providing a way to control cellapoptosis in a cell culture of a specific cell type by the use ofspecific factors for non apoptotic conditions such as a specificcytokine or growth factor or a mixture thereof. Such apoptosis modelsare for example the control of IL-4 stimulated T-cell lines, IL3 andhematopoietic progenitor, PC12 and CRH (corticotropin-releasinghormone), HN9.10.

A candidate compound may modulate apoptosis by blocking or preventingthe association of said pro or anti-apoptotic polypeptide with lipidrafts. In this context, the subcellular localisation of said pro oranti-apoptotic polypeptide in lipid rafts is no more observed in cellsincubated with the compound when compared to cells not incubated withthe compound, or, at least, lipid rafts subcellular localisation of saidpro- or anti-apoptotic polypeptide is significantly reduced whencompared to cells not incubated with the compound. It is another objectof the invention to provide compounds capable of modulating associationof a pro- or anti-apoptotic polypeptide with lipid rafts.

Some of these compounds may modulate apoptosis by preventing theassociation of a pro- or anti-apoptotic polypeptide with lipid rafts, bypromoting segregation of a pro- or anti-apoptotic polypeptide from lipidrafts or by promoting disruption of lipid rafts.

Some of these compounds may modulate apoptosis by preventing thesegregation of a pro- or anti-apoptotic polypeptide from lipid rafts, bypromoting the association of a pro- or anti-apoptotic polypeptide withlipid rafts or by promoting constitution of lipid rafts.

It is another object of the invention to provide compounds capable ofmodulating transfer of a pro- or anti-apoptotic polypeptide between alipid raft and another cellular localization.

Some of these compounds may modulate apoptosis by preventing transfer ofa pro- or anti-apoptotic polypeptide from a cellular localization, otherthan a lipid raft, to a lipid raft or from a lipid raft to anothercellular localization.

Some of these compounds may modulate apoptosis by promoting transfer ofa pro- or anti-apoptotic polypeptide from a cellular localization, otherthat a lipid raft, to a lipid raft or from a lipid raft to anothercellular localization.

In a preferred embodiment, the compounds of the invention modulatingapoptosis are capable of modulating the association of a pro-apoptoticprotein of Bcl-2 family, especially Bad, with lipid rafts or transfer ofsaid protein between lipid rafts and another cellular localization.

Some of said compounds may promote apoptosis in a Bad-producing cell bypreventing association of Bad with lipid rafts, by promoting segregationof Bad from lipid rafts or by promoting disruption of lipid rafts.

Some of said compounds may inhibit apoptosis in a Bad-producing cell bypromoting association of Bad with lipid rafts, by preventing segregationof Bad from lipid rafts or by promoting constitution of lipid rafts.

In a particular embodiment, a compound of the invention may inhibitapoptosis of a Bad-producing cell by preventing transfer of Bad tomitochondria after Bad segregation from lipid rafts. Such a compound mayinteract with Bad by means of a motif similar to the lipid raft motifswhich allow association of Bad to lipid rafts.

Lipid rafts subcellular localisation of said pro- or anti-apoptoticpolypeptide can be quantified by any quantitative analysis methodsavailable in the art. A significant reduction of lipid rafts subcellularlocalisation is observed when the level of pro- or anti-apoptoticpolypeptide is reduced to at least 50%, preferably 90% in cellsincubated with the candidate compound or compared to cells not incubatedwith the candidate compound.

A candidate compound may modulate apoptosis by blocking or preventingthe segregation of said pro- or anti-apoptotic polypeptide from lipidrafts. In this context, the subcellular localisation of said pro- oranti-apoptotic polypeptide in lipid rafts of cells cultured underconditions promoting pro- or anti-apoptotic protein segregation (e.g.,apoptotic conditions for pro-apoptotic proteins) remains observed incells incubated with the compound when compared to cells not incubatedwith the compound and lipid rafts subcellular localisation of said pro-or anti-apoptotic polypeptide is significantly maintained when comparedto cells not incubated with the compound.

Lipid rafts subcellular localisation of said pro- or anti-apoptoticpolypeptide can be quantified by any quantitative analysis methodsavailable in the art. A significant maintenance is observed when thelevel of pro- or anti-apoptotic polypeptide is maintained to at least50%, preferably 90% in cells incubated with the candidate compound orcompared to cells not incubated with the candidate compound or comparedto cells not incubated with the candidate compound.

By “determining the absence of lipid rafts”, it is understood that lipidrafts in cells incubated with the candidate compound are not detected,or at least, are detected as traces, or are detected in a significantlyreduced level (i.e., minimum 20% less) with the methods disclosed in thepresent invention as compared with a culture of cells not incubated withthe candidate compound as a control. The proportion of lipid rafts in acell can be compared between the cell cultures using any quantitativeanalysis methods available in the art. A significant reduction of lipidrafts is observed when their proportion is reduced from 20%, preferablyis reduced to at least 50%, preferably 90% in cells incubated with thecandidate compound to the control. For example, by confocal microscopyanalysis, the profile of fluorescence can be quantified by imageanalysis of cells incubated with the candidate compound and cells notincubated with the candidate compound.

Similarly, by “determining the presence of lipid rafts”, it isunderstood that lipid rafts in cells incubated with the candidatecompound are detected in substantially the same amount as compared witha culture of cells not incubated with the candidate compound as acontrol.

Methods to isolate lipid rafts have been described below. Morespecifically, it is possible to isolate lipid rafts from mammaliancells, by cell fractionating over sucrose gradient and immunobloftingsubcellular fractions with markers specific for rafts in order toidentify rafts containing subcellular fractions. The presence or theabsence of lipid rafts can be thus determined in a specific embodimentby the following steps

-   -   i) recovering the cultured cells incubated with said compound        and resuspending said cells in a buffer appropriate for        subcellular fractionation, such as gradient sucrose buffer;    -   ii) ultracentrifugating the fractionated cells;    -   iii) recovering the subcellular fraction which should contain        lipid rafts; and,    -   iv) determining whether the recovered subcellular fraction        contains ganglioside and/or lipid raft associated molecule(s).

As used herein, “the subcellular fraction which should contain lipidrafts” is the subcellular fraction corresponding to the bandedorganelles of the gradient which contains lipid rafts in a gradientobtained with cells cultured under non apoptotic conditions. Naturally,in apoptotic conditions, the corresponding cell fraction will containmuch less lipid rafts. Lipid rafts and lipid rafts subcellularlocalisation of pro- or anti-apoptotic polypeptides can also be directlyvisualized in intact cells by confocal microscopy using a molecularmarker which specifically binds to a raft-associated molecule or aganglioside.

Such preferred molecular markers are, for example, the cholera toxinsubunit B (CTx) which specifically recognizes ganglioside GM1, anti-Badantibody or anti-Lck antibody. More generally, any antibody directed toany cellular polypeptide newly identified according to the method of theinvention as described above can be used as a molecular marker specificfor raft.

The invention also concerns the compounds identified by the methods ofscreening.

Such compounds identified by the above methods of the invention areuseful for the prevention and/or treatment of disorders induced by orassociated with a defect;He regulation of cell death or of specificpathologies where death of infected or deregulated cells may be at leastpart of a therapy.

The invention further provides a use of a compound capable of modulatinglipid rafts formation, in the preparation of a medicine for thetreatment of disorders induced by or associated with a defectiveregulation of cell death.

In a preferred embodiment, said defective regulation affects cells whichproduce Bad protein, more preferably, cells of the immune system andmost preferably T-cells.

When said defective regulation of cell death results in an abnormaldecrease of cell death, the used compound is preferably a compound whichis capable of disrupting lipid rafts, thereby promoting apoptosis. Suchcompounds are for example, methyl-β-cyclodextrin or filipin. Examples ofdisorders resulting in an abnormal decrease of cell death are cancerdiseases and especially lymphoproliferative cancers, infectious diseasesand especially viral diseases, inflammatory diseases or auto-immunediseases.

Conversely, when defective regulation of apoptosis results in anabnormal increase of cell death, the used compound is preferably acompound capable of reconstituting lipid rafts in the plasma membrane ofcells, such as edelfosine thereby preventing apoptosis. Examples ofdisorders resulting in an abnormal increase of cell death is diseasesassociated to senescence, neuro-degenerative diseases, includingAlzheimer disease, ischemic cell death, wound-healing or AIDS.

The invention provides new means to detect early events of the apoptoticprocess. In particular, the invention enables to identify the apoptoticstate of a cell by determining the presence or the absence of lipidrafts. Accordingly, another object of the invention is an in vitromethod for the detection of a defective regulation of apoptosis, in asample of cells of an individual, said method comprising determining thepresence or the absence of lipid rafts in said cells, wherein theabsence of said lipid rafts is indicative of a defective regulation ofapoptosis.

Examples of methods for determining the presence or absence of lipidrafts in cells have already been described above. In a preferredembodiment, the presence or the absence of lipid rafts is determined bydetecting the presence or absence of a pro-apoptotic or ananti-apoptotic protein which is known to be located in lipid rafts underproliferative growth conditions, such as the Bad protein, or any otherprotein, and especially a cellular polypeptide identified according tothe method of the invention exposed above.

In a specific embodiment, said isolated cells are cells characteristicof the immune system of an individual affected by a lymphoproliferativedisease.

Naturally, the invention also concerns a use of a compound appropriatefor detecting the presence of lipid rafts, in the in vitro detectionmethod described above.

Examples of a compound appropriate for detecting the presence of lipidrafts is a compound which specifically recognizes Bad protein, Lckprotein or ganglioside GM1. In a specific embodiment, said compound usedin the in vitro detection method is selected among cholera toxin subunitB (CTx), anti-Bad antibody and anti-Lck antibody.

LEGENDS TO THE FIGURES

FIG. 1. Effect of IL4 on association of Bad to 14-3-3 protein

Cytoplasmic extracts from 10×10⁶ IL-4-stimulated or -deprived cells wereimmunoprecipitated with anti-Bad or anti-Raf antibodies and blotted withanti-14-3-3, anti-Raf and anti-Bad. Total extracts (lane T) were used asa positive control of 14-3-3 and Rat expression. Similar results wereobtained in three independent experiments.

FIG. 2. Subcellular localization of Bad in IL4-stimulated or -deprivedcells.

A) Anti-Bad, anti-Lck (rafts), CTx-Biotin (GM1 ganglioside, rafts),anti-caspase 3 (cytosol), anti-calnexin (endoplasmic reticulum, ER) andanti-cytochrome C (mitochondria) immunoblot analysis of subcellularfractions from IL4-stimulated or -deprived cells. The fractions (1 to 4)were prepared by sucrose gradient ultracentrfugation and tested fortheir purity using antibodies against mitochondria, rafts, ER andcytosol. Nuclear fraction is not shown in the blot (fraction 5). Proteinloaded per well in each gradient fraction corresponds to that of 5×106cells. Total extracts, 30 μg of protein. Similar results were obtainedin three independent experiments. B) IL-4-stimulated or -deprived cellswere Triton X-100 extracted and fractionated in Optiprep flotationgradient. Fractions were collected from the top to the bottom ofgradient and analyzed by western blot. Only the first, insolubleproteins (I) and the last fraction, soluble proteins (S) are shown.Similar results were obtained in two independent experiments.

FIG. 3. Rafts localization of Bad in IL-4-stimulated cells

A) IL-4-stimulated or -deprived cells were stained with CTX-FITC andeither anti-Lck or anti-Bad antibodies as indicated, followed byCy3-labeled secondary antibody and analyzed by confocal microscopy.

Similar results were obtained in three independent experiments. Singleconfocal sections show fluorescence in green (FITC) and red (Cy3). B)IL-4-stimulated or -deprived cells were stained with anti-Bad andanti-mitochondria antibodies, followed by FITC- and Cy3-labeledsecondary antibodies and analyzed as above. Similar results wereobtained in three independent experiments.

FIG. 4. Methyl-β-cyclodextrin (M-β-CD) treatment abolishes associationof Bad to rafts and induces apoptosis.

A) IL-4-stimulated cells were serum-starved for 30 min and then treatedwith or without 10 mM M-β-CD for 30 min at 370° C. before incubationwith CTx-FITC and anti-Bad or anti-Lck antibodies, followed byCy3-labeled secondary antibody. Then, cells were analyzed by confocalmicroscopy.

Similar results were obtained in two independent experiments. Singleconfocal sections show green (FITC) and red Cy3) fluorescence. B)IL-4-stimulated cells were serum-starved for 30 min and then treatedwith or without 10 mM M-β-CD for 30 min at 37° C., then washed andtransferred to complete medium supplemented with IL-4. At differenttimes, apoptosis was measured. Sub G1 region of the fluorescence scalewas used to determine the percentage of cells present in the initialstep of apoptosis. Similar results were obtained in two independentexperiments. White bars, control cells; grey bars, M-β-CD-treated cells.

FIG. 5. Effect of IL-4 on serine phosphorylation of Bad.

A) Cytoplasmic extracts from IL-4-stimulated or -deprived cells wereimmunoprecipitated with anti-Bad antibody and blotted with anti-Badserine 136, 112 and 155. As internal control, the blot was developedwith anti-Bad. Positive control for serine 112 and 136 phosphorylation,IL-2-stimulated 1; cells; positive control for serine 155phosphorylation of Bad, Bad-transfected COS cells (C). B) Western blotfrom FIG. 2A was proved with anti-Bad serine 136 antibody. Molecularweight of the corresponding proteins is shown.

EXAMPLES 1. Materials and Methods

1.1 Cells, Lymphokines and Reagents

TS1αβis a murine T cell line that can be propagated independently inIL-2, IL-4 or IL-9 Cells were cultured in RPMI-1640 as previouslydescribed (Pitton et al., 1993, Cytokine 5, 362-371). Murine rIL-4 orsupernatant of a HeLa subline transfected with PKCRIL-4.neo was used asa source of murine IL-4. Fluorescein isothiocyanate (FITC-)-labeledcholera toxin (CTx) B subunit, CTx-Biotin and methyl-p-cyclodextrin(M-β-CD) were obtained from Sigma-Aldrich (St. Louis, MO). Cy3- andCy2-conjugated secondary antibodies were purchased from Molecular Probes(Eugene, OR). Anti-mitochondria serum (mito 2813, pyruvatedehydrogenase) was a gift from Dr A. Serrano (CNB, Madrid, Spain).

1.2 Immunoprecipitation and Western Blot

Cells (1×107) were IL-4-stimulated or -deprived and lysed for 20 min at4° C. in lysis buffer (50 mM Tris-HCl pH 8, 1% Nonidet P-40, 1137 mMNaCl, 1 mM MgCl₂, 1 mM CaCl₂, 10% glycerol and protease inhibitormixture). Lysates were immunoprecipitated with the correspondingantibody (Calbiochem Transduction Laboratory). Protein A-Sepharose wasadded for 1 h at 4° C. and, after washing, immunoprecipitates wereseparated by SDS-PAGE. Alternatively, cells were lysed in Laemmli samplebuffer and protein extracts separated by SDS-PAGE, transferred tonitrocellulose, blocked with 5% non fat dry milk in Tris-buffered saline(TBS, 20 mM Tris HCl pH 7.5, 150 mM NaCl) and incubated with primaryantibody in TBS/0.5% non fat dry milk. Membranes were washed with 0.05%Tween 20 in TBS and incubated with PO-conjugated secondary antibody.After washing, proteins were developed using the ECL system.

1.3 Cell Cycle Analysis

A total of 2×10⁵ IL-4-stimulated cells treated with or without M-β-CDwere washed, resuspended in PBS, permeabilized with 0.1% Nonidet P-40and stained with 50 μg/ml propidium iodide (PI). At different times,samples were analyzed using a EpicsXL flow cytometer (Coulter, Hialeah,Fla.). Apoptosis was measured as the percentage of cells in the sub-G₁region of the fluorescence scale having an hypodiploid DNA content.

Cell cycle was also analyzed by annexin staining. A total of 2×10³ cellswere washed with ice-cold PBS diluted in ice-cold binding buffer andstained with annexin and propidium iodide. Samples were maintained onice for 10 min in the dark and then analyzed by flow cytometry.

1.4 Subcellular Fractionation

Subcellular fractionation was performed as previously described (Hackiet al., 2000, Oncogene 19, 2286-2295; Millan and Alonso, 1998, Eur. J.Immunol. 28, 3675-3684). Briefly, IL-4-stimulated or -deprived cellswere washed in PBS and then resuspended for 2 min in extractionbuffer-STE (10 mM Hepes pH 7.4, 1 mM EDTA, 0.25 mM sucrose, 2 μg/mlaprotinin, 10 μg/ml leupeptin, 1 mM PMSF, 1 μg/ml pepstatin). Theextract was inspected under the microscope and more than 95% of thecells were lysed. The homogenates were applied to a linear gradientsucrose (0.73 to 1.9M) and ultracentrifuged at 20,000 g overnight. Thebanded organelles were recovered by syringe, diluted with an equalvolume of 10 mM Hepes buffer and sedimented at the speed appropriatedfor the respective organelles. The purity of the organelles wasdetermined by Western blot using antibodies against specific markers:anti-cytochrome C for mitochondria, anti-Lck and CTx-Biotin for rafts,anti-calnexin for endoplasmic reticulum (ER) and anti-caspase 3 forcytosol. For preparation of cytosol, the homogenate was precentrifugedat 750 g for 10 min to remove nuclei and unbroken cells, followed by acentrifugation a 100,000 g for 1 h to clear off the membranes.

1.5 CTx-FITC Labeling

IL-4-stimulated or -deprived cells were fixed with 1% paraformaldehydefor 5 min on ice, perneabilized and then incubated with CTx-FITC (20min, 6 μg/ml) and anti-Bad antibody for 1 h in PBS-BSA. Cy3-labeledsecondary antibody was added and incubated for 1 h. Finally, and afterseveral washing steps, cells were incubated with methanol at −20° C. for10 min, mounted with Vectpshield medium, and analyzed by confocalmicroscopy. The program used for quantification of samples was Leica TSCNT version 1.5.451 (Leica, Lasertechnik, Heidelberg, Germany).

1.6 Cholesterol Depletion

IL-4-stimulated serum-deprived cells were treated for 30 min at 37° C.with 10 mM M-β-CD, washed and then incubated with CTx-FITC and anti-Bador anti-Lck antibodies as above. Secondary antibody was added andincubated for 1 h. Finally, cells were incubated with methanol at −20°C. for 10 min and mounted as described above.

1.7 Triton X-100 Flotation

IL-4-stimulated or deprived cells were lysed in TXNE buffer (50 mM TrisHCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.2% Triton X-100) containingprotease inhibitor mixture. Detergent insoluble membranes were isolatedby ultracentrifugation (17,000 g, 4 h, 4° C.) in 30-35% gradient ofOptiprep as previously described (Manes et al., 1999, EMBO J. 18,6211-6220).

1.8 Isolation of Mitochondria and S-100 Fraction

Mitochondria were isolated using a modification of the method describedby Yang et al., 1997, Science 275: 1129. Briefly, 20×106 cells were IL-4stimulated or deprived, harvested, and washed with ice-cold PBS. Cellpellet was suspended in 5 vol. icecold buffer A (20 Mm HEPES-KOH (pH7.5), OmM KCl, 1,5 MM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mMPMSF, and 250 mM sucrose) supplemented with protease inhibitors. Cellswere disrupted in a Dounce homogenizer (Kontes, Vineland, NJ), thenucleic were centrifuged (1.000×g, 10 min. 4° C.), and the supernatantwas further centrifuged (1.000×g. 15 min., 4° C.). The resultingmitochondrial pellet was resuspended in buffer A and stored at −80° C.The supernatant was centrifuged (100,000×g, 1 h, 4° C.), and theresulting S-100 fraction was stored at −80° C.

2. RESULTS

2.1 Bad Associates with Lipid Rafts in IL-4-stimulated Cells

It has been shown that after IL-3-stimulation, Bad becomesphosphorylated, resulting in association to 14-3-3 protein. Morerecently, it has been shown that IL-2 induces Bad phosphorylation, butnot association with 14-3-3 protein (Ayllbn et al., 2001, J. Immunol.166, 7345-7352). FIG. 1 shows that neither IL-4-stimulation norIL-4-deprivation results in association of Bad to 14-3-3 protein. Asinternal control, the interaction of Raf and the 14-3-3 protein is shown(FIG. 1).

The subcellular distribution of Bad in IL-4-stimulated or -deprivedcells has been analyzed. IL-4-stimulated or -deprived cells were lysedand fractionated over sucrose gradient. To validate the gradientprotocol, fractions (1 to 4) were immunoblotted with markers for rafts(Lck and GM1 ganglioside), mitochondria (cytochrome C), endoplasmicreticulum (calnexin) and cytosol (caspase 3). Nuclear fraction (fraction5) is not shown in the blot because there is not Bad localization in thenucleus. Rafts were detected by western blot in fraction 1 using antiLckantibody and CTx-Biotin, which recognizes GM1 ganglioside (FIG. 2A).Most of Bad was found in rafts (fraction 1), although a very smallfraction was also present in mitochondria (fraction 4), cytosol andendoplasmic reticulum (fraction 2). As internal control, Bad wasobserved in total extracts of IL-4-stimulated cells. Finally, it hasbeen observed that the fraction of Bad that is sequestered in lipidrafts is dephosphorylated.

It has been previously reported that Bcl-2 is expressed inIL-2-stimulated cells and BCl-X_(L) in IL-4 cultured cells (Gomez, J. etal, 1998, Oncogene 17: 1233). When IL-4-maintained cells are deprived oflymphokine, they undergo apoptosis. As early as 4 h after IL-4deprivation, ≈9% of the cells were apoptotic, reaching 40% at 24 h,whereas control IL-4-stimulated cells showed no significant level ofapoptosis.

IL-4-deprivation induces disorganization of rafts (fraction 1), whichare not detected using either anti-Lck antibody or CTx-Biotin. Themitochondria marker, which also contains other cellular structures withsimilar density, is observed in fraction 4 and most of the caspase 3 iscleaved, given a new protein of lower molecular weight. Moreinteresting, Bad is almost undetectable in cytosol and rafts are onlyobserved in fraction 4, which corresponds to mitochondria and cellularstructures with similar density (FIG. 2A). This result strongly suggestsan IL-4-dependent association of Bad with rafts and translocation tomitochondria upon IL-4-deprivation. Rafts were also isolated by TritonX-100 flotation gradient. As shown in FIG. 2B, Bad and Lck are detectedin the detergent insoluble fraction (I) of IL-4-stimulated cells, whichcorresponds to lipid rafts. In IL-4-deprived cells, Bad and Lck aredetected in the fraction corresponding to soluble proteins (S). It hasbeen observed that post-translational myristoylation targets Bad torafts (data not shown).

The subcellular localization of Bad was also analyzed in mitochondrialand cytosolic fractions of IL-4-stimulated or deprived cells. Bad wasdetected in the mitochondrial fraction of IL-4-stimulated cells. Theamount of Bad associated with mitochondra increased upon IL-4deprivation. Traces of Bad were detected in the cytosolic fraction ofIL4 stimulated or Deprived cells. The antiapoptotic molecule BCl-x_(L)was weakly detected in the mitochondrial fraction of IL-4-stimulatedcells, increasing after IL-4 deprivation. As an internal control ofprotein fractionation, the blot was probed with anti-caspase 3(cytosolic marker), anti-mitochondria Mito 2813 (pyruvate dehydrogenase,mitochondrial marker), and anti-calnexin to show the lack of endoplasmicreticulum contamination in mitochondrial preparation. Total extracts(late T) were used as a positive control of calnexin expression.Finally, the association of Bad with some Bcl-2 family members wasexplored. Coimmunoprecipitation experiments of cytoplasmic proteinsunder IL-4 stimulation or deprivation conditions using specificantibodies were performed. Bad was detected by Western blot inanti-BcI-x_(L) immunoprecipitates of IL-4 stimulated cells, decreasingthroughout the starvation period analyzed. Probing the membrane withanti-Bcl-x_(L) antibodies showed similar levels in all analyzedconditions.

Bad association to rafts in IL-4-stimulated cells was also analyzed inintact cells by confocal microscopy (FIG. 3A). IL-4-stimulated or-deprived cells were incubated with the raft marker cholera toxin Bsubunit (CTx-FITC) before secondary labelling with anti-Bad or anti-Lckantibody. Double immunofluorescence analysis with anti-Bad and CTx-FITCshowed raft localization of Bad in the surface of IL-4-stimulated cells.In marked contrast, a disorganization of rafts in IL-4-deprved cells wasobserved and consequently, no rafts localization of Bad in IL-4-deprivedcells (FIG. 3A). Double immunofluorescence analysis with anti-Lck andCTx-FITC was used as a positive control of localization of Lck inmembrane rafts of IL-4-stimulated cells. Lck associated with rafts wasnot detected in IL-4-deprived cells (FIG. 3A).

The profile of green and red fluorescence colocalization was analyzedusing the quantification software of Leica (TCS NT; Leica, Rockleigh,N.J.). A high number of green and red colocalization peaks was observedin the membrane of IL-4 stimulated cells stained with CTx-Lck orCTx-Bad. On the contrary, the level of colocalization of green and redfluorescence was strongly reduced in IL-4-deprived cells.

This result suggests that Bad is preferentially localized in lipid raftsin IL-4-stimulated cells and segregates from plasma membrane inIL-4-deprived cells.

Similar results of colocalization of Bad with lipid rafts were observedusing freshly isolated thymocytes from mice.

Bad association with mitochondria in IL-4-deprived cells was alsoanalyzed in intact cells by confocal microscopy (FIG. 3B). Doubleimmunofluorescence analysis with anti-Bad and anti-mitochondriaantibodies shows weak association of Bad to mitochondria inIL-4-stimulated cells while there is a high fraction of Bad associatedto mitochondria in IL-4-deprived cells (FIG. 3B). This separation of Badfrom rafts correlates with its translocation to mitochondria inIL-4-deprived cells, as shown by cellular fractionation and confocalmicroscopy (FIG. 2A and 2B).

The profile of green and red fluorescence colocalization was alsoanalyzed using quantification software (Leica) and showed moderate greenand red colocalization peaks in IL-4-stimulated cells stained withanti-Bad and anti-mitochondria Abs. The level of colocalization of bothfluorescences strongly increased in IL-4 deprived cells.

2.2 Association of Bad to Lipid Rafts is Required for Prevention ofApoptosis

Depletion of cellular cholesterol impairs the ability of glycosylphosphatidylinositol (GPI)-anchored proteins to associate with lipidrafts. To examine whether there is a similar requirement of cholesterolfor the association of Bad with rafts, IL-4-stimulated cells weretreated for 30 min with or without 10 mM methyl-β-cyclodextrin (M-β-CD)in serum-free medium to deplete cellular cholesterol. Cells were thenincubated with CTx-FITC and labeled with anti-Bad or anti-Lckantibodies. Serum depletion alone weakly disrupt the association of Lckor Bad to lipid rafts (FIG. 4A). However, M-β-CD treatment causes asevere disruption of raft formation and association of Lck and Bad withrafts in IL-4-stimulated cells (FIG. 4A). This result indicates thatdisruption of raft formation by cholesterol depletion inducessegregation of Bad and Lck from rafts in IL-4-stimulated cells.

Given that exclusion of Bad from rafts was also observed in apoptoticIL-4-deprived cells (FIG. 3A), it was analyzed whether Bad associationto rafts and its integrity was necessary for prevention of apoptosis.For this purpose, IL-4-stimulated cells were treated for 30 min with orwithout M-β-CD in serum-free medium, then washed, resuspended inIL-4-supplemented complete medium and analyzed for induction ofapoptosis at different times (FIG. 4B). M-β-CD treated cells showedstronger level of apoptosis compared with control non treated cells,reaching the highest level 5 hours after M-β-CD treatment. Eight hoursupon treatment, the amount of apoptotic cells detected in treated andnon treated cells were similar because addition of serum restores thelipid composition of the membrane.

This result suggests that segregation of Bad from rafts is involved inthe induction of apoptosis. Posttranslational modifications of Bad suchas phosphorylation, and its role in Bad localization in rafts ormitochondria was further analyzed. FIG. 5A shows that IL-4 inducesserine 136 phosphorylation of Bad, but not serine 112 and 155. Moreover,IL-4-deprivation induces serine 136 dephosphorylation of Bad. Given thatIL-4 induces serine 136 phosphorylation of Bad, western blot wasreprobed from FIG. 2A with anti-Bad serine 136 antibody. FIG. 5B showsthat while most of Bad is localized in rafts in IL-4-stimulated cells,only the weak cytosolic fraction of Bad is serine 136 phosphorylated. InIL-4-deprived cells, traces of serine 136 phosphorylation are observedin cytosol and mitochondria. This result suggests that dephosphorylatedBad is sequestered in rafts and IL-4-deprivation induces segregation andtranslocation to mitochondra.

Subcellular localization of Bad enables to discover how Bad function maybe regulated by dynamic interaction with lipid rafts or mitochondra.

The distinct Bad distribution and function is directly related toIL-4-stimulation or -deprivation of the cells.

These data show that 14-3-3 protein does not control the proapoptoticrole of Bad, contrary to previous reports. On the basis of this result,the subcellular distribution of Bad in IL-4-stimulated or -deprivedcells was analyzed. These results show that different plasma membranefractions can be separated using subcellular fractionation sucroseultracentrifugation gradient because raft markers were successfullyresolved from non-rafts markers. Rafts and mitochondria were alsoisolated by Triton X-100 flotation gradient and differentialcentrifugations, respectively. There are precedents for reversible raftassociation as has been shown following the movement of singlefluorescence lipid molecules (Schutz et al., 2000, EMBO J. 19, 892-901).In addition, after activation by ligand binding the epidermal growthfactor migrates out of rafts into bulk plasma membrane (Mineo et aL,1999, J. Biol. Chem. 274, 30636-30643). The association of proteins withlipid rafts can be modulated because some proteins may be excluded fromrafts by association to other proteins (Field et al., 1995, Proc. NatI.Acad. Sci. USA. 92, 9201-9205). Association of Bad with rafts may beinvolved in steps leading to Bad inactivation, because rafts do notconstitute the final site of activation. IL-4-deprivation inducessegregation of Bad from rafts. This results suggests a two stepsapoptotic process: first, segregation of Bad from rafts, that triggersapoptosis and second, disorganization of lipid rafts during apoptoticprocess. This is strongly suggested by results showing that disruptionof cholesterol rich rafts prevents Bad association and induces apoptosisin IL-4-stimulated M-β-CD-treated cells. Addition of fetal calf serum toIL-4-supplemented medium restores the lipid components of the plasmamembrane, preventing progression of apoptosis.

Localization of proteins to distinct subcellular fractions is anessential step in multiple signaling pathways, including apoptosis.According to this, it has been shown that some signaling molecules aresequestered in rafts. Cholesterol depletion disrupts lipid rafts andmodulates the activity of multiple signaling pathways in T lymphocytes(Kabouridis et al., 2000, Eur. J. Immunol 30, 954-963). These resultsstrongly suggest that in the absence of association of Bad to 14-3-3protein, Bad is sequestered in rafts, avoiding a proapoptotic role andassociation with partners. IL-4 deprivation-induced segregation of Badfrom rafts correlates with translocation to mitochondria and inductionof apoptosis. Restriction of intermolecular interactions bysequestration in lipid rafts has been also described from the α-chain ofthe IL-2R, avoiding its association with the β-and γ-chains of the IL-2R(Marmor, M; and M. Julius, 2001, Blood 98:1489). It is interesting tonotice that in IL-4-stimulated cells, most of cellular Bad localizes inrafts in a dephosphorylated condition while the weak cytosolic fractionis serine 136 phosphorylated. These results show for the first time thesequestration of a proapoptotic protein into lipid rafts as a mechanismthat controls the availability of said proapoptotic protein.

1-8. (canceled)
 9. A method of screening compounds for their capacity tomodulate apoptosis in cells which produce pro- or anti-apoptoticpolypeptides which are located in lipid rafts when said cells arecultured under non apoptotic conditions, said method comprising: a.culturing said cells in a growth medium for maintaining non apoptoticconditions; b. contacting said cultured cells with a candidate compound;c. determining the level of one or several pro- or anti-apoptoticpolypeptides associated to lipid rafts ; and, d. selecting the compoundwhich interferes with the association of one or several pro- oranti-apoptotic polypeptides with lipid rafts, said compound having thecapacity to modulate apoptosis.
 10. A method of screening compounds fortheir capacity to promote apoptosis in cells, said method comprising a.culturing mammalian cells in a growth medium for maintaining nonapoptotic conditions; wherein said cells produce a pro-apoptotic proteinwhich is located in lipid rafts under non apoptotic conditions of saidcells; b. contacting said cultured cells with a candidate compound; and,c. determining the absence or the presence of lipid rafts in saidcultured cells; d. in case of presence of lipid rafts, optionallydetermining the level of pro-apoptotic protein located in the lipidrafts, wherein the absence of lipid rafts in the plasma membrane of,cells incubated with said candidate compound or if determined, thereduced level of pro-apoptotic protein in the rafts is indicative thatsaid compound promotes apoptosis.
 11. A method of screening compoundsfor their capacity to inhibit or prevent apoptosis of cells, said methodcomprising a. culturing cells in a growth medium for maintaining nonapoptotic conditions; wherein said cells produce a pro-apoptotic proteinwhich is located in lipid rafts under, non apoptotic conditions; b.contacting said cells with a candidate compound; c. culturing cellsunder apoptotic conditions; d. determining the absence or the presenceof lipid rafts; e. in the case of presence of lipid rafts, optionallydetermining the level of pro-apoptotic protein located in the lipidrafts; wherein the presence of lipid rafts in the plasma membranes ofcells incubated with said candidate compound and optionally themaintained level of pro-apoptotic protein in the rafts is indicativethat said candidate compound inhibits or prevents apoptosis.
 12. Themethod according to claim 9, wherein a pro-apoptotic protein located inlipid rafts under proliferative conditions is the Bad protein.
 13. Themethod of claim 12, wherein said cells which produce a Bad protein arecells characteristic of the immune system, preferably T cells.
 14. Themethod according to claim 9, wherein the presence or the absence oflipid rafts is visualized by confocal microscopy.
 15. The methodaccording to claim 9, wherein the presence or the absence of lipid raftsis determined by the following steps i) recovering the cultured cellsincubated with said compound candidate and resuspending said cells in abuffer appropriate for subcellular fractionation, such as gradientsucrose buffer; ii) ultracentrifugating the fractionated cells and; iii)recovering the subcellular fraction which should contain lipid rafts;iv) determining whether the recovered subcellular fraction containsganglioside and/or lipid raft associated molecule(s).
 16. The methodaccording to claim 15, wherein the presence or the absence of lipidrafts is determined by the use of a marker which specifically recognizesganglioside or a raft-associated molecule.
 17. The method according toclaim 16, wherein said marker is selected among cholera toxin subunit B(CTx), anti-Bad antibody or anti-Lck antibody.
 18. The method of claim9, wherein said cells are mammalian cells.
 19. The method of claim 9,wherein said non apoptotic conditions are proliferative conditions. 20.The method of claim 9, wherein said growth medium comprises at least acytokine or a growth factor necessary for maintaining proliferativegrowth conditions.
 21. The method of claim 11, wherein the apoptoticconditions of step c) are obtained by depriving the cells with saidcytokine or growth factor.
 22. The method of claim 20, wherein acytokine or growth factor necessary for maintaining proliferative growthconditions is an interleukin, preferably selected among IL-4, IL-2 orIL-9, or a mixture thereof.
 23. A use of a compound capable ofmodulating lipid rafts formation, in the preparation of a medicine forthe treatment of disorders induced by or associated with a defectiveregulation of cell death or of any specific pathology in which celldeath may be at least a part of the therapy.
 24. The use of claim 23,wherein said defective regulation affects cells which produce Badprotein.
 25. The use of claim 24, wherein said defective regulationaffects cells of the immune system.
 26. The use of any of claim 23,wherein said compound is capable of disrupting lipid rafts and whereinsaid defective regulation of cell death results in an abnormal decreaseof cell death.
 27. The use of claim 26, wherein said abnormal decreaseof cell death is related to cancer disease and especially tolymphoproliferative cancers, infectious disease and especially viraldisease, inflammatory disease or auto-immune disease.
 28. The use of anyof claim 23, wherein a compound capable of disrupting lipid rafts ismethyl-β-cyclodextrin or filipin.
 29. The use of claim 23, wherein saidcompound is capable of reconstituting lipid rafts in the plasma membraneof cells and wherein said defective regulation of apoptosis results inan abnormal increase of cell death.
 30. The use of claim 29, wherein apathology resulting in an abnormal increase of cell death is a diseaseassociated to senescence, neurodegenerative disease Alzheimer, AIDS,ischemic cell death or wound-healing.
 31. The use of claim 29, wherein acompound capable of reconstituting lipid rafts is edelfosine.
 32. An invitro method for the detection of a defective regulation of apoptosis,in a sample of cells of an individual, said method comprisingdetermining the presence or the absence of lipid rafts in said cells,wherein the absence of said lipid rafts is indicative of a defectiveregulation of apoptosis.
 33. The method according to claim 32, whereinsaid cells are cells of the immune system of an individual affected by alymphoproliferative disease.
 34. The method of claim 32, wherein thepresence or the absence of lipid rafts is determined by detecting thepresence or absence of a pro-apoptotic or an anti-apoptotic proteinwhich is known to be located in lipid rafts under non apoptoticconditions of cells.
 35. The method of claim 34, wherein a pro-apoptoticprotein known to be located in lipid rafts under non apoptoticconditions is a Bad protein.
 36. A use of a compound appropriate fordetecting the presence of lipid rafts, in the in vitro detection methodaccording to claim
 32. 37. The use of claim 36, wherein a compoundappropriate for detecting the presence of lipid rafts is a compoundwhich specifically recognizes Bad protein, Lck protein or gangliosideGM1.
 38. The use of claim 37, wherein said compound is selected amongcholera toxin subunit B (CTx), anti-Bad antibody and anti-Lck antibody.39. A use of a compound capable of modulating pro- or anti-apoptoticprotein rafts localization for the preparation of a medicine for thetreatment of disorders induced by or associated with a defectiveregulation of cell death or of any specific pathology in which celldeath may be at least a part of the therapy.
 40. The use of claim 39,wherein said compound promote pro- or anti-apoptotic protein segregationfrom lipid rafts.
 41. The use of claim 39, wherein said compoundpromotes pro- or anti-apoptotic protein localization in lipid rafts. 42.An in vitro method for the detection of a defective regulation ofapoptosis, in a sample of cells of an individual, said method comprisingdetermining the presence or the absence of lipid rafts in said cells,wherein the presence of a great number of lipid rafts compared to normalcells is indicative of a defect regulation of apoptosis.
 43. The methodaccording to claim 10, wherein a pro-apoptotic protein located in lipidrafts under proliferative conditions is the Bad protein.
 44. The methodof claim 43, wherein said cells which produce a Bad protein are cellscharacteristic of the immune system, preferably T cells.
 45. The methodaccording to claim 10, wherein the presence or the absence of lipidrafts is visualized by confocal microscopy.
 46. The method according toclaim 10, wherein the presence or the absence of lipid rafts isdetermined by the following steps i) recovering the cultured cellsincubated with said compound candidate and resuspending said cells in abuffer appropriate for subcellular fractionation, such as gradientsucrose buffer; ii) ultracentrifugating the fractionated cells and; iii)recovering the subcellular fraction which should contain lipid rafts;iv) determining whether the recovered subcellular fraction containsganglioside and/or lipid raft associated molecule(s).
 47. The methodaccording to claim 45, wherein the presence or the absence of lipidrafts is determined by the use of a marker which specifically recognizesganglioside or a raft-associated molecule.
 48. The method according toclaim 47, wherein said marker is selected among cholera toxin subunit B(CTx), anti-Bad antibody or anti-Lck antibody.
 49. The method of claim10, wherein said cells are mammalian cells.
 50. The method of claim 10,wherein said non apoptotic conditions are proliferative conditions. 51.The method of claim 10, wherein said growth medium comprises at least acytokine or a growth factor necessary for maintaining proliferativegrowth conditions.
 52. The method according to claim 11, wherein apro-apoptotic protein located in lipid rafts under proliferativeconditions is the Bad protein.
 53. The method of claim 52, wherein saidcells which produce a Bad protein are cells characteristic of the immunesystem, preferably T cells.
 54. The method according to claim 11,wherein the presence or the absence of lipid rafts is visualized byconfocal microscopy.
 55. The method according to claim 11, wherein thepresence or the absence of lipid rafts is determined by the followingsteps i) recovering the cultured cells incubated with said compoundcandidate and resuspending said cells in a buffer appropriate forsubcellular fractionation, such as gradient sucrose buffer; ii)ultracentrifugating the fractionated cells and; iii) recovering thesubcellular fraction which should contain lipid rafts; iv) determiningwhether the recovered subcellular fraction contains ganglioside and/orlipid raft associated molecule(s).
 56. The method according to claim 54,wherein the presence or the absence of lipid rafts is determined by theuse of a marker which specifically recognizes ganglioside or araft-associated molecule.
 57. The method according to claim 56, whereinsaid marker is selected among cholera toxin subunit B (CTx), anti-Badantibody or anti-Lck antibody.
 58. The method of claim 11, wherein saidcells are mammalian cells.
 59. The method of claim 11, wherein said nonapoptotic conditions are proliferative conditions.
 60. The method ofclaim 11, wherein said growth medium comprises at least a cytokine or agrowth factor necessary for maintaining proliferative growth conditions.